We have examined the ability of cytotoxic lymphocytes to killing of mammalian red blood cells as simple target cells. Using crosslinked hybrid antibodies against the T cell receptor and a target membrane antigen, efficient lysis of both red cell and nucleated targets could be achieved using cloned CTL. Splenic T cells or thymocytes do not cause detectable target lysis. Surprisingly, cloned helper T cells also killed red cell targets but not nucleated targets when triggered by this method. The physiology of red cell lysis by both effectors appears to be similar to classical CTL-mediated killing of nucleated targets. The mechanism of lysis by LGL granule cytolysin as been analyzed by taking advantage of the observation that both low pH and low ionic strength (in isotonic sucrose) block lysis at a step after cytolysin binding to target cells. After washing off all unbound cytolysin a stable intermediate is formed which will lyse when resuspended in a buffer containing isotonic saline at neutral pH. Calcium is required for this lytic step by either route of intermediate formation. The binding step requires calcium in low ionic strength, but not at low pH. The bound cytolysin can be digested off by proteases or neutralized by anti-cytolysin antibodies. When nucleated tumor cells are exposed to cytolysin at sub-lethal doses, they show evidence of membrane injury as indicated by their rapid membrane depolarization and loss of ions and small markers. However, this sublethal injury is repaired by the tumor cells within a few minutes, as shown by the restoration of membrane potential and normal distribution of sodium and potassium. We have found that polystyrene beads of diameter 3-15 mu coated with both anti-T3 and anti-target antibody will trigger CTL to kill cells not normally recognized. This lysis appears to be a special case of bystander lysis, which is classically not observed in CTL cytotoxicity. We have developed a new general method for measurement of cytotoxicity using target labeling by the fluorescent dye BCECF. This should replace the standard 51Cr release method for many purposes.